After mating, behavioral and physiological changes are seen in the female insect. Compared to virgins, mated Drosophila melanogaster females are largely unreceptive to further mating, lay eggs at an elevated rate, live less long, and store and efficiently utilize sperm (reviewed in Hall, 1994; Chen, 1996; Kubli, 1996; Wolfner, 1997). These changes in the female occur because she receives, via seminal fluid, secretions from her mate's accessory gland and also sperm (see Chen, 1996; Kubli, 1996; Wolfner, 1997 for reviews and for original references). Products of the predominant cell type of the accessory gland, the main cells, are necessary for changes in the female's egg-laying rate and receptivity on the first day after mating. Stored sperm cause these effects to persist for up to 11 days following mating. Accessory gland main cell secretions also shorten the life span of the mated female. In addition, they play a role in the storage of sperm and in the competition between sperm from sequential matings.
Knowledge of how accessory gland products mediate these changes is important in understanding the control of insect fertility and the mechanisms of peptide hormone action. Once genes encoding Accessory gland proteins (Acps) are identified, genetic and molecular genetic techniques uniquely possible in Drosophila can be used to dissect the role of each protein in reproduction. In a few cases, it is possible to identify the functions of Acps by injecting purified fractions into unmated female flies and observing behavioral effects. For example, in D. melanogaster, a “sex peptide” (“SP”) of 36 amino acids was purified and shown to stimulate egg-laying and depress receptivity to mating for one day (Chen, 1988). SP was cloned and shown to derive from a single gene at chromosomal position 70A (Chen, 1988). A sex peptide and a second peptide, ovulation-stimulating substance (OSS), with similar activities have also been purified from D. suzukii (Ohashi, 1991; Schmidt, 1993).
Only Acps which can be purified or synthesized in active forms, act on their own and act via the hemolymph, can be identified by such assays. In order to identify Acp genes without presupposition of function, differential cDNA hybridization can be used to isolate RNAs expressed only in accessory glands (Schäfer, 1986; DiBenedetto, 1987; Monsma and Wolfner, 1988). cDNA hybridization screens are more likely to isolate abundant RNAs in a tissue. Thus, they are biased towards RNAs expressed in main cells of the accessory gland (96% of the secretory cells of the accessory gland; Bertram, 1992) rather than the rarer secondary cells (4% of the secretory cells of the gland; Bertram, 1992). Previous differential cDNA hybridization screens for genomic clones encoding male-specific transcripts identified three genomic regions encoding Acps (Schäfer, 1986; DiBenedetto, 1987). Of these, the 95EF region encodes a small secreted Acp (DiBenedetto, 1990), 57D contains a gene cluster encoding three small peptides (Simmerl, 1995), and the 51F locus has not yet been characterized. In addition to these genes, a region encoding two Acps has been identified by screening a “chromosomal walk” for accessory gland-specific transcription units (Monsma and Wolfner, 1988). In this region, only 20 bases separate the gene for Acp26Aa, an ELH-similar prohormone-like molecule (Monsma and Wolfner, 1988) that stimulates egg-laying in the mated female fly (Herndon and Wolfner, 1995), from the gene for Acp26Ab, a small peptide of as yet unknown function (Monsma and Wolfner, 1988). The previously-isolated Acp genes are only a small subset of Acp genes, though the total number of Acp genes is difficult to estimate from prior protein electrophoretic data (e.g. Ingman-Baker and Candido, 1980; Stumm-Zollinger and Chen, 1985; Whalen and Wilson, 1986; Coulthart and Singh, 1988) as summarized and discussed in Chen (1991). This is because on the one hand in the electrophoretic studies small peptides were not resolved, while on the other hand some Acps run as multiple bands on SDS gels (Monsma and Wolfner, 1988). To gain a more complete picture of the spectrum of proteins produced by the accessory gland, a differential screen aimed directly at accessory gland-specific RNAs was performed.